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Sterol 14alpha-demethylase (P45051) is the target for azole antifungal compounds, and resistance to these drugs and agrochemicals is of significant practical importance. We undertook site-directed mutagenesis of the Candida albicans P45051 heterologously expressed in Saccharomyces cerevisiae to probe a model structure for the enzyme. The change T315A reduced enzyme activity 2-fold as predicted for the removal of the residue that formed a hydrogen bond with the 3-OH of the sterol substrate and helped to locate it in the active site. This alteration perturbed the heme environment, causing an altered reduced carbon monoxide difference spectrum with a maximum at 445 nm. The changes also reduced the affinity of the enzyme for the azole antifungals ketoconazole and fluconazole and after expression induced by galactose caused 4-5-fold azole resistance in transformants of S. cerevisiae. This is the first example of a single base change in the target enzyme conferring resistance to azoles through reduced azole affinity.

Two Cryptococcus neoformans strains isolated from an AIDS patient were investigated, a pretreatment isolate (CN1) and a second isolate (CN3) following failure of fluconazole and amphotericin B treatment. No difference in fluconazole sensitivity, but relative resistance to amphotericin B was observed for CN3. The sterol composition of CN3 indicated a defect in sterol delta 8-->7 isomerase in this strain and depletion of ergosterol, the major sterol of the CN1.

Cytochrome P-45061 (CYP61) was a cytochrome P-450 revealed during the yeast genome project when chromosome XIII was sequenced. Here we report on the properties of this second microsomal P-450 of vegetatively growing yeast. The enzyme kinetics associated with its endogenous role in sterol ⌬ 22-desaturation revealed a K m of 20.4 M and a V max of 2.9nmol/min/nmol CYP61. The affinity of the enzyme for antifungal drugs was characterized to investigate its potential role in determining tolerance to these sterol 14␣-demethylase (CYP51) inhibitors. Drug binding induced a type II spectral change, which became saturated at equimolar concentrations of azole drug and P-450. Fluconazole exhibited slightly reduced affinity in comparison to ketoconazole as indicated by carbon monoxide displacement. These and K i determination for fluconazole (0.14 nM) revealed CYP61 to have a similar affinity to azole drugs when compared with data available for CYP51, and the implications for antifungal treatment were considered.The cytochrome P-450 (CYP) superfamily are involved in a variety of monooxygenase reactions including xenobiotic and endogenous substrates. Genome projects are uncovering more genes encoding such proteins, and one such instance was cyp61 (ERG5) encoded on chromosome XIII of Saccharomyces cerevisiae (1). Previous studies on S. cerevisiae have identified a single form of P-450 during purification from microsomal fractions of vegetatively growing yeast. This P-450 performed a role in sterol 14␣-demethylation (2), later associated with the gene, cyp51 (ERG11; Ref. 3). This is the only P-450 activity associated with a family found in plants, animals, and fungi (4). Other studies based on inhibition and co-factor characteristics indicated a further P-450-mediated activity was present in vegetative yeast (5). This was confirmed when such a protein was purified from microsomes obtained from a vegetatively growing strain with a cyp51 gene disruption (6). N-terminal amino acid sequence confirmed the protein to be CYP61 from chromosome XIII (1).Inhibitors of CYP51 are of considerable commercial importance as antifungal compounds and selectively inhibit fungal CYP51 over the mammalian and plant counterparts (7). Because they may also bind to CYP61, a potential antifungal target, we characterized the affinity of this P-450 for two of the main drugs employed, ketoconazole and fluconazole, to consider the relative potency and potential contribution CYP61 may make to azole antifungal susceptibility. EXPERIMENTAL PROCEDURESMaterials-Unless specified, all chemicals were obtained from Sigma (Poole, Dorset, UK). Ketoconazole was purchased from Janssen Pharmaceutica, and fluconazole was from Pfizer. Microsomes were prepared, and CYP61 was purified from semi-anaerobically grown cells of the S. cerevisiae strain DK2, which contains a gene disruption in cyp51 (⌬cyp51; ⌬erg11) as described previously (6). Rabbit NADPH-cytochrome P-450 reductase was a gift from Prof. M. Akhtar, University of Southampton. Ergosta-5,7-dienol was purified from a ...

: The mechanism of resistance to ICIA 5504 (azoxystrobin) in a Septoria tritici mutant raised in the laboratory has been investigated. This mutant was approximately 10 times less sensitive than the wild-type strain in in-vitro tests towards spore germination or fungal growth. Glucose oxidation in whole cells was inhibited in the wild type (80% inhibition at 0É1 kg ml~1), whereas in the resistant mutant, oxygen uptake was stimulated (50% stimulation at 1É0 kg ml~1). Respiration of the wild-type strain was inhibited by antimycin A and cyanide but not that of the mutant. These results indicate the existence of an efficient alternative respiratory pathway in the mutant, which was inhibited by the addition of 2 mM salicylhydroxamate (SHAM). Using mitochondria, antimycin A and ICIA 5504 did not completely inhibit NADH oxidation in either strain. Addition of SHAM inhibited part of the antimycin-and ICIA 5504-insensitive oxygen uptake only in mutant mitochondria. For complete inhibition of oxygen reduction, SHAM and cyanide need to be present. Thus, three systems of electron transfer from exogenous NADH to oxygen are present in S. tritici mitochondria : the cytochrome pathway which is sensitive to ICIA 5504 and antimycin A inhibition in both strains, the system of NADH-cytochrome c reductase which bypasses the methoxyacrylate inhibition at the cytochrome complex, bc 1 and the alternative oxidase which is inhibited by SHAM, and which is partially functioning only in mitochondria isolated from the ICIA 5504-resistant mutant.When the S. tritici isolates were tested for their in-vivo sensitivity to ICIA 5504 on wheat, the resistant strain was controlled better than the wild type. This indicates that the decreased ATP formation by the alternative pathway of respiration was inadequate for efficient parasitic growth on the host.